Microcontact Printing of Proteins for Cell Biology

Micro contact printing is used to pattern proteins and other molecules on material surfaces to recreate the complexity of extracellular environments. To begin micro contact printing, a topological master is created and then soft lithography is used to cast a stamp from the master out of Syl guard. The stamp can be coated with protein and then used to stamp proteins on a new surface on which cells can adhere.

Hi, my name is Chen. I'm from Dr.Lance Camp's lab in biomedical engineering Columbia University. Hi, I'm GT from the Mechanical Engineering Department.

Hello, I'm Lance Kem from the Department of Biomedical Engineering at Columbia University. We use this procedure in our laboratory to study how cells respond to complex arrangements of proteins in their extracellular environment. This was developed in the mid 1990s by the laboratory of George Whitesides and has become a widely used and valuable tool.

There are many variations to this technique, but today G and Co U will be demonstrating the basic micro contact printing process. So let's get started. For micro contact printing, you should prepare the cover slips and protein solution for stamping several days in advance.

First, immerse the cover slips into the limbo solution for 10 minutes heating and stirring until the solution is clear. This process as well as the following steps for cleaning and storage can be simplified by loading cover slips into ceramic staining racks. Next extensively, rinse the cover slips with deionized water bake the rinse cover slips at 45 degrees Celsius for six hours.

To prepare the protein solution, reconstitute fibronectin following the manufacturer's instructions to a stock of one milligram per milliliter concentration. These stocks are stored frozen at negative 20 degrees Celsius. With working aliquots in temporary storage at four degrees Celsius.

Casting stamps can be carried out several days in advance of the experiment. These stamps can be stored pattern up in a covered dish like a tissue culture dish. To begin, first, remove loose dust from the master using a stream of compressed filtered air or inert gas.

Next, place the master pattern side up in the bottom of a plastic dish, which is slightly larger than the master and should be coated in aluminum foil. 60 or 100 millimeter tissue culture dishes work well for this purpose. In the polystyrene 50 milliliter centrifuge tube, combine the cyl guard components at a ratio of curing agent to elastomer base of one to 10 by weight.

Mix thoroughly with a disposable plastic instrument like a pipette tip. Prepare enough of the elastomer such that there is at least 0.2 milliliters of elastomer per square centimeter of dish area. Centrifuge the 50 milliliter tube at 300 G for five minutes.

To remove air bubbles after centrifugation or the elastomer over the master, then place under vacuum such as inside a desiccate for 30 minutes. Next, cure the elastomer in an oven at 65 degrees Celsius for at least two hours. Curing at higher temperatures and for longer times results in stiffer elastomer.

After curing, let the master cool to room temperature. Finally, separate the sheet of stamps from the master and then trim stamp to the dimensions of the master to micro contact. Print fiber nin onto the glass cover slips first.

Cut out a single stamp stamps measuring four millimeter by four millimeter to one centimeter by one centimeter in area and one to two millimeter thick are easiest to start with. Place the stamp pattern side up on a glass slide or small plastic dish. Next, place the stamp in a plasma cleaner and process under a vacuum for 30 seconds.

A herrick scientific plasma cleaner set at its highest output setting will render the PDMS surface hydrophilic. If this is set for longer times, it will result in cracking of the elastomer. Dilute the fiber nin solution with PBS to a stamping concentration of 50 micrograms per milliliter.

Once the solution is ready, place a small drop of about 10 to 50 microliters of the stamping solution on the stamp. The solution will spread across the hydrophilic surface. Add only enough solutions so that the drop covers the stamp, but does not run over the edges.

Let protein absorb to the stamp for five minutes. Afterwards, use a Kim wipe or other clean paper tissue to wick off most of the protein solution from the stamp without touching the patterned region. Dry the remaining solution from the stamp under a stream of clean, dry inert gas such as nitrogen.

Once dry, use tweezers to remove the stamp from the glass slide invert and place the stamp in contact with the cleaned glass cover. Slip the surface to be patterned. Place a weight on top of the stamp to promote good contact.

The specific weight that provides the best patterning is dependent on the stamp size and pattern. Start with the five gram weight and adjust between stampings. Leave the stamp in contact with the surface for one minute.

Once finished, carefully disassemble the stack and separate the stamp from the cover Slip vigorously. Rinse the patterned cover slip in PBS followed by deionized water to remove protein that is not absorbed to the surface dry the cover slip under a nitrogen stream. Micro contact printing is a powerful process for creating patterns of molecules on surfaces.

In this example, we pattern fluorescently labeled fibronectin in the pattern of our school logo. Each logo measures 200 micrometers in height. A powerful aspect of this process is that multiple rounds of micro contact printing can be combined on a single surface, as in this example of two different antibodies overlaid on a single surface.

In this case, the green spots measure one micrometer in diameter and are spaced at four micrometer intervals. We use these surfaces to investigate how cells integrate multiple cues of the extracellular environment. In this movie, naive T cells are seated onto an array of spots containing antibodies to cell surface receptors.

Cells locate and interact with these features, self-organizing irregular arrays. The micro contact printing process is conceptually simple and very robust, having been applied to patterning a wide range of molecules on a variety of substrates. However, this process remains something of an art.

The specific geometry of the pattern to be created, protein to be patterned applied weight and coating stamping conditions all affect the stamping quality. We demonstrate the patterning of fibronectin on the glass as a reliable starting point for learning these techniques. We have just shown you the basics of micro contact stamping when doing this procedure.

Always remember to follow the lab safety precautions. So that's it. Thank You for watching and good luck with your experiments.